Lipase enzymes are known to catalyze hydrolysis of triglycerides and other fats in accordance with the following reaction: ##STR1##
The lipase enzyme hydrolysis reaction requires water as a reactant, as indicated above. Lipase enzyme is a hydrophilic protein, and water is its natural environment, so that lipase enzyme-catalyzed reactions normally are carried out in an aqueous medium.
Recently, it has been found that some enzymes, among them lipases, also hydrolyze fats in organic solvent media. Water-miscible solvents, such as ethanol and acetone, cannot be used because of their denaturating effect on the enzymes. For example, aqueous alcohol solutions are used for wound treatment, because of their denaturating effect. However, hydrocarbons such as hexane and petroleum ether are effective in lipase-catalyzed hydrolysis, because the enzymes are stable in such solvents, but water as a reactant must be present, although the amount of water can be quite small. If the solvent system is anhydrous, i.e., if no water is present, the lipase catalyzes ester formation, i.e., the reverse reaction between fatty acid and glycerol to form a triglyceride, but not the hydrolysis of the triglyceride to form fatty acid and glycerol. Thus, it is possible simply by controlling the amount of water present to carry out either hydrolysis or condensation, using lipase enzymes. The water need not even be present in a water phase, or distributed in the organic solvent, but can also be hydrated or absorbed on a sorbent such as Celite or calcium carbonate, which is then dispersed in the organic solvent.
The fact that enzymes can function in what is essentially an anhydrous environment containing little or no water has expanded the range of utilization of enzyme-catalyzed reactions in organic synthesis. Both hydrolyses and condensations can be carried out, and solubility problems with organic substrates in water can be avoided, since organic solvents can be substituted for the water.
The use of lipases in condensation reactions of fatty acids with glycerol is of no practical interest, Because the triglycerides are available as raw materials in any desired quantities, and are inexpensive. However, transesterification of naturally-occurring triglycerides, or triglycerides prepared from naturally-occurring fats and oils, is of interest, because this enables the substitution of any desired combination of fatty acid groups in selected positions on the triglyceride. It is accordingly possible, starting from inexpensive naturally-occurring or prepared triglycerides, to prepare by transesterification triglycerides substituted with fatty acid groups in combinations that do not exist or are rarely found in nature. Since many lipase enzymes are selective in the positions which they attack in transesterification reactions on the triglyceride molecule, it is possible to incorporate the desired fatty acid groups in any selected position on the triglyceride molecule, i.e. positions 1, 2, or 3, or a random distribution of all three.
K. Yokozeki et al, Europ J. Appl Microbiol Biotechnol 14 1 (1982); T. Tanaka et al, Agric Biol Chem, 45 2387 (1981); and M. H. Coleman and A. R. Macrae, U.S. Pat. No. 4,275,081, patented June 30, 1981, describe the transesterification of triglycerides using position-specific lipases such as Rhizopus delemar to convert inexpensive triglyceride distillation fractions derived from palm oil and olive oil to a fatty acid composition and distribution which corresponds to that of cocoa butter. Natural cocoa butter is in very short supply, and quite expensive.
The chemical reaction in this transesterification can be illustrated as follows: ##STR2## in which S is stearic acid, P is palmitic acid, and O is oleic acid. A mixture of stearic acid and a triglyceride with palmitic acid residues in positions 1 and 3 and an oleic acid residue in position 2 has been converted into a mixture of palmitic acid and a new triglyceride where the stearic acid residue now is in positions 1 and 3. The 1,3-specific lipase enzyme catalyzes a transesterification reaction in positions 1 and 3, and leaves position 2 intact.
Of course, other reactions take place as well, because palm oil or olive oil is not a pure P--O--P triglyceride, and cocoa butter is not a pure S--O--P triglyceride, but the above reaction does constitute a meaningful representation of what primarily takes place.
The process as described in the literature quoted above is preferably carried out in hexane or other aliphatic hydrocarbon. However, the reaction rate is slow, and reaction times of the order of 40 to 50 hours are required, to bring the transesterification to completion. The reaction temperature is effectively limited to 40.degree. C., because of enzyme instability at higher temperatures, and so it is not possible to overcome the slow reaction rate by increasing the reaction temperature. Thus, the process has not been commercialized, because such long reaction times make the process uneconomic.